PROJECT SUMMARY Radiation exposure from a large-scale nuclear incident could have catastrophic consequences. Significant morbidity and mortality may result from damage to the vascular endothelium. Endothelial cells are central to all organs, and radiation-induced endovascular injury may result in both acute and delayed organ toxicity, such as acute lung injury, pulmonary fibrosis, and impaired hematopoiesis. These outcomes result from several pathological effects on the endothelium, which begin with endothelial barrier disruption and vascular leak. Endothelial integrity and barrier function are regulated in large part by the endothelial receptor tyrosine kinase Tie2. Tie2 activation by its agonist ligand, angiopoietin-1 (Ang-1), promotes vascular integrity, preventing vascular leak induced by inflammation. In contrast, Ang-2, a Tie2 antagonist, inhibits the stabilizing effects of Ang-1, thereby promoting vascular leak in a variety of pathological conditions, such as sepsis and chemical- induced lung injury. Importantly, Ang-2 expression is increased in endothelial cells after exposure to radiation, suggesting that decreased Tie2 activity is an important factor in radiation-induced endovascular injury. In addition to Ang-2, Tie2 is negatively regulated by vascular endothelial-protein tyrosine phosphatase (VE-PTP). Our group has tested a highly selective small molecule inhibitor of VE-PTP, AKB-9785, which acts as a pharmacological Tie2 activator, and shown that it promotes endothelial barrier function in preclinical models. By targeting this pathway, we may increase Tie2 activity, prevent vascular leak after radiation, and improve outcomes. In addition to Tie2, platelets also regulate endothelial barrier function by occupying gaps in the endothelial lining; releasing soluble factors (including Ang-1) to enhance barrier function; promoting the growth of endothelial cells; and maintaining the endothelial ultrastructure. Paucity of platelets (i.e., radiation-induced thrombocytopenia) may mediate vascular leak and other downstream effects. Though thrombocytopenia may be addressed via transfusion, donor platelets are limited in supply and may not be available in a mass radiation event. To meet this need, we have developed fibrinogen-coated nanoparticles (FCN) as a novel therapeutic strategy. Imaging studies suggest that FCN bind to endothelial cells, serving a physical presence (i.e., plugging gaps), and recruit the remaining platelets to sites of need. Thus, FCN may have both direct effects (improved hemostasis) and indirect effects (Ang-1/Tie2 activation) on the endothelium, and preliminary studies in murine models of radiation-induced thrombocytopenia suggest that FCN improve survival. While AKB-9785 and FCN may appear to target different pathways, the role of Ang-1 is an important point of overlap. Therefore, we propose to further characterize the acute and delayed effects of radiation on endothelial cells in the lung and bone marrow, focusing on the endothelial Tie2 signaling pathway and thrombocytopenia as likely mediators of radiation damage. We also propose study to novel countermeasures (AKB-9785, FCN) targeting these pathways to mitigate the effects of radiation-induced endothelial injury.